Coherently controlled nonlinear raman spectroscopy and microscopy

ABSTRACT

A method and system are presented for producing an output coherent anti-stokes Raman scattering (CARS) signal of a medium. The method comprises generation of a unitary optical excitation pulse that carries a pump photon, a Stokes photon and a probe photon; and inducing a CARS process in the medium by exciting the medium by the at least one such unitary optical excitation pulse.

FIELD OF THE INVENTION

The present invention relates to Raman spectroscopy and microscopy, andin particular, to coherent anti-stokes Raman spectroscopy andmicroscopy.

BACKGROUND OF THE INVENTION

In coherent nonlinear spectroscopy, the sample is probed by measuringprocesses of energy exchange between photons interacting with thesample. One of the most common nonlinear spectroscopy methods iscoherent anti-stokes Raman scattering (CARS), a coherent four-wavemixing process involving the generation of a coherent vibration in theprobed medium. In CARS, three laser photons, a pump photon (ω_(p)) aprobe photon (ω_(pr)) and the Stokes photon (ω_(s)), overlap in themedium under investigation. By nonlinear interaction with the moleculesa fourth coherent photon (ω_(AS)) with the anti-Stokes frequencyω_(AS)=ω_(p)−ω_(s)+ω_(pr) is generated.

The CARS process can be visualized in a molecular energy level diagramas depicted in FIG. 1, where |i

and |β

are molecular rovibrational states, and |α

and |β

are virtual levels. Resonant enhancement of the CARS process occurs whenthe frequency difference Ω_(R)=ω_(p)−ω_(s) coincides with a vibrationallevel of the medium.

The CARS process, as a coherent scattering process, has to fulfill aphase matching condition, which is equivalent to momentum conservationof the photons involved. With the wave vectors of the pump photon(k_(P)), the probe photon (k_(pr)) and the Stokes photon (k_(S)), thewave vector of the Raman signal can be obtained byk _(AS) =k _(pr)+(k _(P) −k _(S)) or k _(P) +k _(pr) =k _(AS) +k _(S).

In general, there are two conventional different techniques utilizing amulti-beam excitation scheme for measuring a CARS spectrum, asdisclosed, for example, in U.S. Pat. Nos. 4,077,719; 4,084,100;4,405,237; and 4,512,660; and in WO 02/48660.

According to the first technique, the so-called scanning CARS, twonarrow bandwidth lasers at ω_(p) and at ω_(s) (having spectral width ofthe order of the typical linewidth of Raman levels, i.e., 1 cm⁻¹) aretuned over the Raman resonances of the probed species to generate asignal at 2ω_(p)−ω_(s) (in this case ω_(pr)=ω_(s)). The spectralresolution of this technique is mainly determined by the bandwidth ofthe applied laser sources.

According to the second technique, broadband or multiplex CARS, abroadband Stokes beam (spectral width typical 100-1000 cm⁻¹) can be usedto excite several Raman transitions under investigations simultaneously.The use of a narrow band probe and a broadband Stokes beam enablessimultaneous measurement of the entire band of the Raman spectrum (see,for example, “Infrared and Raman Spectroscopy,” edited by B. Schrader,VCH, Weinheim, 1995). The spectral resolution of this technique isusually achieved by using a monochromator and a multichannel detectionsystem. Thus, one laser shot is utilized to measure the entire CARSspectrum.

Another possibility to obtain multiplex CARS spectra is to use atime-resolved CARS scheme. In this technique, two relatively broadbandexciting pulses are used for simultaneously populating several Ramanlevels. The spectral data is obtained by measuring the interferencepattern of the CARS signal from a third, delayed broadband probe pulse(see, for example, an article of Leonhardt et al., published in Chem.Phys. Lett., 1987, V. 133, P. 373).

Coherent Raman processes have become a valuable tool in the past fewdecades in femtosecond time-resolved spectroscopy, as well as incombustion studies and condensed-state spectroscopy. For example,Leonhardt et aL describes in Chem. Phys. Lett. 1987, V. 133, P. 373 themeasurements of the energy difference and the lifetimes of two (or more)Raman levels by Fourier-decomposing the quantum beats of the CARS signalusing femtosecond pulses. This scheme has been recently used to analyzethe energy-level diagram of complex molecules.

CARS has recently become a favorable technique for nonlineardepth-resolved microscopy (see, for example, U.S. Pat. No. 6,108,081; WO02/06778; and scientific articles Zumbusch et al., Phys. Rev. Lett.,1999, V. 82, P. 4142; Hashimoto et al., Opt. Lett., 2000, V. 25, P.1768; and Volkmer at al., Applied Phys. Lett., 2002, V. 80, P. 1505).CARS microscopy has the potential, for example, for studying livebiological specimens while gathering three-dimensional information ontheir molecular constitution. However, the these CARS microscopes alsorequire two or three narrow-band sources that must be all tightlysynchronized and also tunable within the Raman energy range.

It should be appreciated that the signal of CARS (being a result of anonlinear process) is stronger with short intense pulses. However, thefemtosecond CARS techniques suffer from two major difficulties. First,there is an increased strong background signal typically due to theelectronic contributions to the third-order susceptibility, both fromthe sample and from the surrounding medium (i.e., solvent). The seconddifficulty is associated with a lack of selectivity between neighboringenergy levels, due to the large bandwidth of the pulses.

These problems can be solved by coherent quantum control methods. Theconcept of coherent quantum control of a quantum system is based on theachievement of constructive interference between different quantum pathsleading to a desirable outcome, while interfering destructively withpaths leading to other outcomes. While schemes of coherent control mayinvolve excitations by continuous waves, most available techniques arealso known which involve ultrashort optical pulses. With the recentprogress in ultrafast optics, it is now possible to shape ultrashortsignals with desired spectral shapes (see, for example, U.S. Pat. No.6,327,068 assigned to the assignee of the present application).

The inventors of the present invention have recently shown how coherentcontrol techniques can be exploited to improve the CARS spectroscopyemploying three femtosecond pulses related to the pump, Stokes and probebeams, respectively. Two approaches have been described for controllingthe CARS process. According to the first approach (“Quantum Control ofCoherent anti-Stokes Raman Processes” by Oron et al., published in Phys.Rev. A, 2002, V 65, P. 43408), a periodic phase modulation is used tocontrol the population induced by broadband pulses. By shaping both thepump and the Stokes pulses with an appropriate spectral phase function,the nonresonant CARS background has been greatly reduced. This techniquealso allows for exciting just one out of many vibrational levels, evenwhen all of them are within the spectral bandwidth of the excitationpulses. According to the second approach (“Narrow-Band CoherentAnti-Stokes Raman Signals from Broad-Band Pulses” by Oron et al.,published in Phys. Rev. Lett., 2002, V 88, P. 63004), only the probepulse is shaped, thereby enabling enhancement of the resolution of themeasured CARS spectrum. The achieved spectral resolution becomessignificantly better than the bandwidth of the readout pulse. Inparticular, by tailoring the phase of a 100 femtosecond probe pulse, anarrow-band CARS spectroscopy resonant signal has been obtained with awidth of less than 15 cm⁻¹, which is an order of magnitude narrower thanthe CARS signal from an unshaped, transform limited pulse (all frequencycomponents having the same phase).

SUMMARY OF THE INVENTION

There is a need in the art to facilitate coherent anti-stokes Ramanscattering (CARS) spectroscopy and microscopy by providing a novelmethod and system for producing an exciting signal to induce a CARSprocess in a medium.

The main idea of the present invention consists of inducing a CARSprocess in a medium (i.e., providing a CARS spectrum of the medium) byexciting the medium with a single pulse carrying a pump photon, a Stokesphoton and a probe photon. In other words, the technique of the presentinvention provides for supplying three interacting photons (the pumpphoton, Stokes photon and probe photon) by the same unitary excitationpulse. This enables the system operation with a single laser sourcegenerating a transform limited femtosecond pulse. The present inventionprovides various coherent-control techniques consisting of shaping thetransform limited pulse broadband pulse (carrying a pump photon, aStokes photon and a probe photon) to produce a unitary opticalexcitation pulse enabling identification of a CARS signal induced bythis pulse from any other optical signal.

The present invention provides for designing a single-pulse CARSspectrometer or microscope free of the two aforementioned difficulties,and for achieving high spectral resolutions and diminishing thedetrimental effects of the nonresonant background.

The concept of the present invention for performing a nonlinear opticalinteraction with a matter in a single coherently controlled pulse offersa promising alternative to the conventional multi-beam nonlinear systemsin use today.

Thus, according to one aspect of the present invention, there isprovided a method for producing an output coherent anti-stokes Ramanscattering (CARS) signal of a medium, the method comprising: (i)producing a unitary optical excitation pulse that carries a pump photon,a Stokes photon and a probe photon; and (ii) inducing a CARS process inthe medium by exciting the medium by the at least one unitary opticalexcitation pulse.

The unitary optical excitation pulse carrying the pump, Stokes and probephotons is produced by generating a transform limited optical pulsecarrying the pump, Stokes and probe photons; and applying apredetermined shaping to the transform limited optical pulse.

The shaping of the transform limited optical pulse may comprise blockingwavelengths shorter than a predetermined wavelength in said pulse. Thispredetermined wavelength is defined by a spectral bandwidth in which theoutput CARS signal is likely to occur.

The shaping of the transform limited optical pulse may compriseassigning a desired phase to each wavelength component of the transformlimited optical pulse. The assigning of the desired phase is preferablycarried out is addition to the blocking of wavelengths shorter than thepredetermined wavelength. The assigning of the desired phase preferablyincludes modulating a spectral phase of the transform limited opticalpulse by using a desired spectral phase function. The desired spectralphase function may be a periodic function, or may be formed by at leastone phase gate having a bandwidth substantially narrower than thebandwidth of the unitary excitation pulse to be produced. The phase gatemay for example be a π phase gate, e.g., with the bandwidth in the rangeof about 0.5 nm to 3 nm. The π phase gate is preferably spectrallylocated in the vicinity of a short wavelength end of the excitationpulse to be produced.

The above shaping can be implemented by passing the transform limitedpulse through a Spatial Light Modulator (SLM).

Alternatively or additionally to the phase modulation, the shaping maycomprise application of polarization control to the transform limitedpulse consisting of 90 degree polarization rotation of predeterminedwavelengths of the pulse. This results in that the inp0ut transformlimited pulse is split into a broadband pump component and a narrow-bandprobe component having substantially orthogonal polarizations.

According to another aspect of the invention, there is provided a pulsecreation method for use in coherent anti-stokes Raman scattering (CARS)spectroscopy or microscopy, the method comprising: utilizing a singlelaser operable to generate a transform limited optical pulse carrying apump photon, a Stokes photon and a probe photon, and applying apredetermined shaping to the transform limited optical pulse to producea unitary optical excitation pulse.

According to yet another aspect of the invention, there is provided amethod for coherent anti-stokes Raman scattering (CARS) spectroscopy ofa medium constituted of molecules capable of producing an output CARSsignal, comprising:

(a) producing at least one unitary optical excitation pulse that carriesa pump photon, a Stokes photon and a probe photon;

(b) focusing said at least one unitary optical excitation pulse onto themedium, thereby exciting the medium to produce the output CARS signal ofthe molecules; and

(c) measuring said output CARS signal.

According to yet another aspect of the invention, there is provided amethod for coherent anti-stokes Raman scattering (CARS) microscopy of atarget material constituted of molecules producing an output CARSsignal, the method comprising:

-   -   producing at least one unitary optical excitation pulse that        carries a pump photon, a Stokes photon and a probe photon;    -   focusing said at least one unitary optical excitation pulse onto        the medium, thereby exciting the medium to produce the output        CARS signal of the molecules;    -   providing a relative displacement between the medium and the        exciting beam to thereby enable scanning of the medium by the        unitary excitation pulse beam.

The invention according to its yet another aspect provides a system foruse in measuring an output coherent anti-stokes Raman scattering (CARS)signal of a medium, the system comprising a single laser operable togenerate at least one transform limited optical pulse carrying a pumpphoton, a Stokes photon and a probe photon, and a programmable pulseshaper for receiving the transform limited optical pulse and shaping itto produce a unitary optical excitation pulse.

There has thus been outlined, rather broadly, the more importantfeatures of the invention in order that the detailed description thereofthat follows hereinafter may be better understood. Additional detailsand advantages of the invention will be set forth in the detaileddescription, and in part will be appreciated from the description, ormay be learned by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carriedout in practice, a preferred embodiment will now be described, by way ofnon-limiting example only, with reference to the accompanying drawings,in which:

FIG. 1 is an energy level diagram of the typical CARS process;

FIG. 2A illustrates a schematic view of a single-pulse CARS spectrometersystem, according to one embodiment of the invention;

FIG. 2B illustrates a schematic view of a single-pulse CARS spectrometersystem, according to another embodiment of the invention;

FIG. 3A to FIG. 3C illustrate effect of a modulated spectral phasefunction on the temporal shape of the pulse and on the populationamplitude;

FIG. 4A to FIG. 4C illustrate examples of measurements of the Ramanspectra obtained by varying the phase function periodicity;

FIG. 5A to FIG. 5B illustrate the nonresonant background suppression byusing periodic spectral function with additional harmonics;

FIG. 6A to FIG. 6C illustrate effect of a π phase gate phase function onthe temporal shape of the pulse and on the population amplitude;

FIG. 7A to FIG. 7D are examples of a numerical simulation illustratingthe effect of the phase control by using an excitation phase with anarrow-band phase gate.

FIG. 8A and FIG. 8B illustrate measured normalized CARS spectra by usinga transform limited pulse a phase gate shaped pulse for methanol andiodomethane, correspondingly;

FIG. 9A to FIG. 9D illustrate the normalized spectral intensity derivedfrom the measured CARS spectra along with those obtained by computersimulations for several materials.

FIG. 10A to FIG. 10D illustrate CARS spectra from iodomethane obtainedwith polarization-only shaping and plotted for various probe bandwidths;

FIG. 11 shows a schematic drawing of the spectral intensity of a phaseand polarization shaped excitation pulse;

FIG. 12 is a schematic drawing of the electric field envelope versustime for phase and polarization shaped pulses;

FIG. 13A to FIG. 13C illustrate CARS spectra from iodomethane obtainedwith both polarization and phase gate shaping and plotted for variousprobe bandwidths;

FIG. 14A to FIG. 14C illustrate examples of Raman spectra of severalsimple molecules obtained with phase and polarization shaped pulses;

FIG. 15 illustrates a schematic view of a single-pulse CARS microscopysystem, according to one embodiment of the invention; and

FIG. 16A to FIG. 16D illustrate an example of depth-resolvedsingle-pulse CARS images.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention provides a method and a CARS system such asspectrometer or microscope carrying out this method based on inducingthe entire CARS process by producing a single (unitary) ultrashortoptical excitation pulse that supplies all three photons (the pumpphoton, Stokes photon and probe photon) required for the CARS process.

The principles and operation of the CARS spectrometry and microscopyaccording to the present invention may be better understood withreference to the drawings and the accompanying description, it beingunderstood that these drawings and examples in the description are givenfor illustrative purposes only and are not meant to be limiting. Thesame reference numerals will be utilized for identifying thosecomponents which are common in the CARS spectrometer and microscopesystems shown in the drawings throughout the present description of theinvention.

It should be noted that the inducing of the entire CARS process by asingle excitation pulse is feasible when the pulse duration is shorterthan the vibrational period of the molecules of the medium underinvestigation. For example, a length of this excitation pulse can be ina femtosecond range. In this case, the CARS signal is produced by anintra-pulse four-wave mixing process. Each of the different componentsof the CARS signal is the resultant of the interference of all thequantum paths that contribute to the nonlinear polarization process.

It should also be noted that inducing the CARS process with a singleexcitation pulse is associated with several inherent difficulties. Firstof all, a technical difficulty arises from the partial spectral overlapbetween the spectral bands of the excitation pulse and the CARS signal,which can be orders of magnitude weaker than the pulse signal.

This difficulty, according to one embodiment of the invention, can beovercame by means of a partial blocking of the excitation pulse spectrumin the range of the expected CARS signal, and an appropriate spectralfiltering of the measured CARS signal.

Moreover, if a medium is excited by a single transform limited pulse(i.e., a pulse in which all frequency components have the same phase),then the CARS process can encounter the following difficulties:

One of the difficulties arises from the fact that all vibrational levelshaving the energy within the bandwidth of the transform limited pulseare excited. As a result, the spectral resolution of the CARS signal islimited by the excitation pulse bandwidth.

Another known difficulty, which is common to all CARS techniquesutilizing femtosecond pulses and is therefore relevant also to thesingle-pulse CARS spectrometer and method of the present invention,results from a strong nonresonant background signal. As the bandwidth ofthe excitation pulse is increased (in other words, as the shorterexcitation pulse having higher peak intensity is used), the magnitude ofthe background signal increases much more rapidly than the resonant CARSsignal. The nonresonant background signal thus can be detrimental to theability to spectrally resolve resonant transitions.

The present invention provides for eliminating the above difficulties byapplying a quantum coherent control technique to the transform limitedoptical pulse. According to the invention, the quantum coherent controlis achieved by means of a predetermined shaping of the transform limitedpulse to produce a unitary optical excitation pulse that enablesidentification of a CARS signal induced by this pulse from any otheroptical signals.

Referring to FIG. 2A, there is schematically illustrated a CARSmeasurement system (spectrometer) 20 according to one embodiment of thepresent invention associated with a sample holder 29 containing a mediumunder investigation. The CARS spectrometer system 20 includes a singlelaser 21 adapted for producing optical transform-limited driving pulses210 wherein each such pulse carries a pump photon, a Stokes photon and aprobe photon which are necessary for exciting the medium and inducingthe CARS process therein, a programmable pulse shaper 22 operable forshaping the input transform limited driving pulse to produce a unitaryoptical excitation pulse carrying the pump, Stokes and probe photons, adetector unit 26 for collecting a CARS signal coming from the medium andgenerating data indicative thereof, and a light directing optics fordirecting the input pulses to the medium and directing the CARS signalto the detector.

The laser 21 can be any laser capable to generate transform limitedpulses in a femtosecond (fs) time range. For example, the transformlimited pulses can be in a range of about 5 fs to 100 fs and,preferably, between 10 fs and 20 fs). For example, a Ti:Sapphire laseroscillator capable of generating 20 fs full-width at half maximum (FWHM)transform limited pulses at 80 MHz, centered at 815 nm (corresponding toa bandwidth of about 75 nm or an energy span of 1100 cm⁻¹) can beemployed for the purpose of the present invention. The programmablepulse shaper 22 is configured for shaping the input transform limiteddriving pulses by assigning a desired phase to each wavelength componentof the transform limited optical pulse, preferably only in apredetermined wavelength range, i.e., outside that where the CARS signalis most likely to occur. The use of such a pulse shaper enables forcoherently controlling the CARS process.

A sample of the input transform limited pulse 210 generated by the laser21 is directed to the pulse shaper assembly 22 by a mirror 23 a, and ashaped pulse 220 produced by the shaper assembly 22 is directed to theholder 29 by a further mirror 23 b. Obviously, each of thesesingle-mirror elements of the light directing optics may be replaced byone or more beam splitter and/or a set of mirrors, or any other knownlight deflecting means.

In the present example of FIG. 2A, the programmable pulse shaper 22 is a4-f shaper that includes an input dispersive assembly 221, an outputdispersive assembly 222; an input focusing element 223, an outputfocusing element 224; and a programmable Spatial Light Modulator (SLM)226 located at the Fourier plane defined by the focusing elements 223and 224. In the present example, also provided in the pulse shaper 22 isa blocking element 225.

For example, the dispersive assemblies 221, 222 can be thin ruledreflective gratings with 1200 lines/mm, and the focusing elements 223,224 can be achromat lenses (e.g., with a focal length of 100 mm). Thoughthe above embodiment uses ruled reflective gratings at the input andoutput of the pulse shaper to spatially disperse and recombine thevarious frequency components of the pulses, it should be pointed outthat any other suitable dispersive elements can be used, e.g.,transmission gratings, prisms, or combinations thereof. Furthermore, thefunction of the focusing elements 223, 224 in defining the systemFourier plane (focal plane) at which the SLM 226 is located, can befulfilled by any other element having positive focusing power, e.g., aconcave mirror.

The blocking element 225 is, for example, a plate arranged for blockingat the Fourier plane wavelengths shorter than a predetermined wavelength(e.g., 780 nm) in the range of a CARS signal, as they can spectrallyoverlap an output CARS signal 211. In the present example of FIG. 2A,the blocking element 225 is arranged between the SLM 226 and the outputfocusing element 224. However, as can be appreciated by a person skilledin the art, the blocking element 225 can be arranged either upstream ordownstream of the SLM 226. Furthermore, the function of blocking element225 can be fulfilled by any sharp-edge long-pass filter, e.g., adielectric filter.

The programmable SLM 226 may be a liquid crystal based SLM of the typedescribed by A. M. Weiner in the article published in Rev. Sci. Inst.,2000, V. 71, P. 1929. This SLM includes an SLM pixel array having 128pixels at its Fourier plane. The spectral resolution, determined by thespot size at the Fourier plane, can be better than 0.5 nm (equivalent toabout 8 cm⁻¹).

The operation of the 4-f pulse shaper 22 is as follows. The inputdispersive element 221 operates to spatially separate the frequencycomponents of the input transform limited pulse. The input focusingelement 223 focuses each of these frequency components to its specificposition at the focal plane, where the SLM 226 is located. The blockingelement 225 blocks at the Fourier plane wavelengths shorter than thepredetermined wavelength. The SLM 226 is operative as an updateablefilter for spectral manipulation of the incoming pulses, and allows theindependent control of the phase and amplitude of each of the lightcomponents passing through 128 pixels, thereby modifying the pulse shapeand temporal profile according to the desired pulse properties. Forexample, the width of each pixel is 97 μm, the inter-pixel gap is 3 μm,while the spot size at the focal plane is about 80 μm. The outputfocusing element 224 and output dispersive element 222 then recombineeach of the separate frequency components to produce a shaped pulse 220.

Thus, the programmable pulse shaper 22 of FIG. 2A is operable by asuitable control unit 27 for separating between different frequencycomponents of the input pulse, blocking the predetermined frequencies(higher frequencies, which can overlap the CARS signal); and assigningthe desired phase to each frequency component of the remaining(non-blocked) portion of the input pulse by using any desired spectralphase function.

The CARS spectrometer system 20 may utilize an open loop control, inwhich the applied spectral phase function is derived theoretically foreach experiment, or may utilize a closed feedback loop for determiningthe applied spectral phase function.

The shaped pulse 220 produced by the pulse shaper 22 carries a pumpphoton, a Stokes photon and a probe photon (which are necessary forexciting the medium and inducing the CARS process therein) and istherefore also referred to as “the unitary optical excitation pulse”.

The unitary optical excitation pulse 220 reflected from the mirror 23 bis focused by a focusing assembly 24 a onto the medium underinvestigation for exciting thereof and inducing a CARS process. In thepresent example, the focusing assembly 24 a includes an objective lensarrangement having an NA=0.2 numerical aperture.

The light directing optics further includes a lens assembly 24 baccommodated for collection of the output CARS signal of the medium.This lens assembly 24 b, preferably, has numerical aperture similar orlarger than that of the lens 24 a. It should be noted that the focusingassembly 24 a can also serve as a collecting optics in a back-scatteredmode.

Further provided in the CARS spectrometer system 20 is a filteringassembly 25 operable for filtering the CARS signal obtained from themedium. In the present example, the filtering assembly 25 includes aspectral filter 251 (e.g., a bandpass or short-pass filter), andpreferably includes a computer-controlled monochromator 252 or aspectrograph (not shown). An example of the filter 251 includes, but isnot limited to, a 40 nm FWHM bandpass filter centered at 750 nm, whilean example of the computer-controlled monochromator 252 includes, but isnot limited to, a computer-controlled monochromator with a spectralresolution of 0.5 nm (equivalent to about 8 cm⁻¹ at 750 nm).

The filtered output of the filtering assembly 25 is collected by thedetector unit 26, which includes a detector 261 of the kind receiving alight signal and generating an electrical output indicative thereof, andmay also include a lock-in amplifier 262 operable by the control unit(computer) 27.

It should be appreciated that the construction and operation of thefilter 251, monochromator 252, detector 261 and a lock-in amplifier 262as well as the elements of the light directing optics, are known per se,and therefore need not be specifically described.

It should be noted that the measurable Raman energy range of the system20 can, for example, be about 300 cm⁻¹-900 cm⁻¹, that is typical ofcarbon-halogen bond stretching. The lower limit of the measurable energyrange is determined by the need to filter out the excitation pulse,while the upper limit is dictated by the excitation pulse bandwidth.Since the technique does not require an electronic resonance with thedriving input field, it can be implemented with any broadband opticsource 21. The measurable Raman energy range of the system 20 can beextended to the fingerprint region (900 cm⁻¹-1500 cm⁻¹) by using pulsesof duration 10 fs-20 fs, available in the state-of-the-art commerciallyavailable lasers.

Referring to FIG. 2B, a schematic view of a CARS spectrometer system 200according to another embodiment of the present invention isschematically illustrated. The CARS spectrometer system 200,distinguishes from the CARS system 20 shown in FIG. 2A in that itsshaper assembly additionally to the phase modulator or as an alternativethereto comprises a polarization control assembly including afrequency-selective filter, such as grating 221, and a 90 degreepolarization rotator accommodated in the optical path of the frequencycomponents emerging from the filter and operable for applying a 90degree polarization rotation to the predetermined frequency range of theinput transform limited laser pulse, and comprises a crossed polarizerunit P_(y) accommodated in the optical path of a signal propagating fromthe medium to the detector for extraction of the cross-polarized CARSsignal.

In the present example of FIG. 2B, the polarization rotator isconstituted by an SLM arrangement 226, but it should be understood thatany other suitable means can be used (e.g., a half-wavelength assembly).Additionally, in the example of FIG. 2B, the polarization controlassembly also includes a polarizer P_(x) accommodated in the opticalpath of the transform limited laser pulse propagating towards the shaperassembly. It should however be understood that the provision of thisinput polarizer P_(x) is optional, and can be eliminated by using alaser source producing linearly polarized light. In the example of FIG.2B, the SLM unit can be operable for performing both the phase shapingand polarization rotation of the frequency components of the transformlimited pulse. Generally, the phase assembly may comprise only apolarization rotator of any known suitable type. The SLM assemblysuitable to be used in the system of the present invention may, forexample, be of the type described by T. Brixner et al. in the articlespublished in Opt. Lett., 2001, V. 26, P. 557 and Appl. Phys., 2002, V.B74, P. S133. Such a programmable liquid crystal SLM 226 includes twoSLM liquid crystal pixel arrays (dual cell SLM) whose preferential axesare at right angles to each other and are rotated by ±45° relative tothe polarization of the input laser pulse (denoted as the x direction).Any difference in the applied retardance between the two arrays resultsin modification of the input pulse polarization. In this technique, theSLM can act as both a controlled spectral phase mask and as a controlledwaveplate.

Thus, in this specific example, the programmable pulse shaper, inoperates for both assigning the desired phase to each frequencycomponent of the driving laser pulse, and a polarization control of thepulse. In particular, the polarization control can be used to break theultrashort input pulse 210 into a broadband pump and a narrow-band probewith orthogonal polarizations.

The nonlinear polarization producing the CARS signal driven by anelectric field of the excitation pulse whose spectrum is E(ω) can beapproximated for nonresonant transitions, by using time dependentperturbation theory, as (for more details see, for example, Oron et al.,Phys. Rev. Lett., 2002, V. 88, P. 63004): $\begin{matrix}{{P_{nr}^{(3)} \propto {\int_{0}^{\infty}\quad{{\mathbb{d}\Omega}\quad{E\left( {\omega - \Omega} \right)}{A(\Omega)}}}},} & (1)\end{matrix}$where A(Ω)=∫₀ ^(∞)dω′E*(ω′−Ω)E(ω′) is the probability amplitude topopulate a vibrational level with energy

Ω (henceforth, the population amplitude), while E(ω−Ω) represents theprobe field.

Similarly, the nonlinear polarization for a singly resonant Ramantransition through an intermediate level |i

at an energy of

Ω_(R) and a bandwidth Γ can be approximated by $\begin{matrix}{P_{r}^{(3)} \propto {\int_{0}^{\infty}\quad{{\mathbb{d}\Omega}\frac{E\left( {\omega - \Omega} \right)}{\left( {\Omega_{R} - \Omega} \right) + {i\quad\Gamma}}{{A(\Omega)}.}}}} & (2)\end{matrix}$

The CARS process can be controlled by controlling the populationamplitude A(Ω). The control of A(Ω) is accomplished by controlling thespectral phase of the single broadband excitation pulse. Such aphase-only pulse shaping of the pulse merely means multiplication of theelectric field E(ω) (that includes pump, Stockes and probe photons) by aphase function exp(iΦ(ω)).

The population of a vibrational level at energy

Ω_(R) is proportional to|A(Ω)|² =|∫dωE(ω)E*(ω−Ω_(R))|²,  (3)where E=|E(ω)|exp(iΦ(ω)) is the complex spectral amplitudes of theapplied field. Each level is thus excited by all frequency pairsseparated by Ω_(R). The interference between the multiple paths leadingto the population of the level Ω_(R) is determined by the relative phaseof each contribution Φ(ω)−Φ(ω−Ω_(R)). Thus, constructive interference isachieved when Φ(ω)=Φ(ω−Ω_(R)) for all frequency components of theexcitation pulse. Therefore, if the excitation pulse is atransform-limited pulse (all frequency components having the samephase), then the constructive interference holds for all values ofΩ_(R), thus spectral resolution is lost.

According to one example of the phase control, that has been firstdescribed by the inventors in an article entitled “Single-pulsecoherently-controlled nonlinear Raman spectroscopy and microscopy”published in Nature, V 418, PP. 512-514 (August 2002), the spectralphase of the excitation pulse is modulated periodically with a periodΩ₀. In such a case, the constructive interference can be induced for allenergy levels only for Ω_(R)=NΩ₀ (where N is an integer). FIGS. 3A-3Cillustrate an effect of a modulated spectral phase function on thetemporal shape of the pulse and on the population amplitude, accordingto this specific example of the phase control.

FIG. 3A shows an example of an input pulse spectral intensity 31, aspectral phase 32 of the transform limited pulse, and a modulatedspectral phase 33 of the shaped pulse (unitary excitation pulse). Alsoshown is a typical spectral region where the CARS signal can bemeasured. Thus, in order to avoid the spectral overlap between the inputpulse and a CARS signal, the power spectrum of the input pulse isblocked at 730 nm. (Note that the spectral intensity of the CARS signalis identified by a reference numeral 34).

FIG. 3B shows in time domain a temporal intensity of transform limitedpulse 35 corresponding to the uniform phase (32 in FIG. 3A), and amodulated phase shaped pulse 36 corresponding to the modulated phase (33in FIG. 3A). As can be understood from FIG. 3B, a periodic spectralphase is equivalent to splitting the pulse in time domain to severalequally spaced pulses, each delayed by τ₀=2π/Ω₀. This pulse train iscapable of resonantly exciting only vibrations with a period T=τ₀/N

FIG. 3C shows a calculated population amplitude A(Ω) for the transformlimited pulse 37 and for the pulse having the modulated phase 38. As canbe seen in FIG. 3C, for the case of the transform limited pulse, thepopulation amplitude decays monotonically versus the vibration energy.On the other hand, for the case of a modulated phase function, anoscillation appears where the peak of each oscillation reaches thetransform limited result.

The resonant and nonresonant processes described above have differentspectral responses, which result from the different weights thatmultiply the population amplitude in Eq. (2) and Eq. (3), as determinedby the resonance levels.

According to Eq. (2), the resonant CARS process can be expressed as$\begin{matrix}\begin{matrix}{P_{r}^{(3)} \approx {C\left\lbrack {{i\quad\pi\quad{E\left( {\omega - \Omega_{R}} \right)}{A\left( \Omega_{R} \right)}} + {{\int_{0}^{\infty}\quad{\mathbb{d}\Omega}}}} \right.}} \\\left. {\frac{\Omega - \Omega_{R}}{\left( {\Omega - \Omega_{R}} \right)^{2} - \Gamma_{R}^{2}}{E\left( {\omega - \Omega} \right)}{A(\Omega)}} \right\rbrack\end{matrix} & (4)\end{matrix}$where ζ is the principal value of Cauchy. The first term in Eq. (4)corresponds to the “on-resonant” contribution, while the second termcorresponds to integration over the contribution of the “off-resonant”spectral components. The resonant signal thus has a narrow responsearound Ω=Ω_(R).

The weight function of the integrand of the second term inverts its signaround the resonance, therefore the total contribution of the integraldepends on the symmetry of E(ω−Ω)A(Ω) around Ω=Ω_(R). In the transformlimited case, both A(Ω) and E(ω−Ω) are nearly symmetric for all valuesof Ω. Therefore, the off-resonant term is negligible. The polarizationspectrum can be approximated in this case byP_(r) ⁽³⁾˜iπCE(ω−Ω_(R))A(Ω_(R))  (5)which is a replica of the pulse spectrum shifted by Ω_(R). Spectralphase manipulation will change the symmetry of E(ω−Ω)A(Ω) for differentvalues ω, and will therefore induce variations of the polarizationspectrum.

However, when the modulation frequency significantly exceeds 2π/Δω(here, Δω is the pulse spectral bandwidth), the total signal intensityaverages out and is proportional to |A(Ω_(R))|².

By the same token, the measured nonresonant intensity averages out tobecome proportional to${{\int{\frac{\mathbb{d}\Omega}{\Omega}{A(\Omega)}}}}^{2}.$The total measured signal is the interference of the signals generatedby the two different processes. In the common case where the nonresonantbackground is considerably larger than the resonant signal, the resonantsignal is measured by a “heterodyne detection” with it, to yield|P ⁽³⁾(ω)|² =|P _(r) ⁽³⁾(ω)+P _(nr) ⁽³⁾(ω)|² ˜P _(r) ⁽³⁾(ω)|²+2Re[P _(r)⁽³⁾(ω)P _(nr) ⁽³⁾(ω)*].  (6)

By exploiting the different spectral response of the resonant andnonresonant components, it is possible to significantly reduce thenonresonant background while maintaining the resonant signal. Followingthe above derivation, it can be clear to a person versed in the art thatproperly choosing the periodicity of the phase function, the populationand thus the resonant CARS signal is reconstructed to nearly the valueachieved by a transform limited pulse.

Thus, it was shown above that the coherent control of the CARS processvia manipulation of the vibrational level population amplitude A(Ω) canbe accomplished by applying periodic spectral phase function (e.g.,sinusoidal function) producing periodical modulation of the spectralphase of the excitation pulse.

Referring to FIGS. 4A-4C, measurements of the Raman spectrum areexemplified. The Raman spectrum has been obtained by monitoring thetotal CARS signal, while varying the phase function periodicity.According to this examples, a simple periodic spectral phase functionΦ(ω)==1.25 cos(Cω) (wherein C is a constant) has been applied to theinput transform limited pulse for its shaping. Presented in FIGS. 4A-4Care the intensities of the CARS signal versus the number of periods ofthe sinusoidal phase across the spatial light modulator (SLM). Theinsets show the Raman spectrum, derived by Fourier transformation of thecorresponding measured intensity signals.

More specifically, FIG. 4A illustrates single-pulse CARS spectroscopy ofmethanol molecules in the liquid phase. As can be seen for methanol,which has no Raman resonance levels at the measured frequency range,only a monotonic decrease of the nonresonant signal is observed as thenumber of oscillation periods is increased.

FIG. 4B illustrates single-pulse CARS spectroscopy of CH₂Br₂ moleculesin the liquid phase. As can be seen, for CH₂Br₂, which has a singleresonance at Ω_(R)=577 cm⁻¹, the Raman signal oscillates periodically,whenever the modulation period is an integer fraction of Ω_(R). TheFourier transform operation retrieves the single Raman resonant levelwith a resolution that is inversely proportional to the number ofmodulation periods.

FIG. 4C illustrates single-pulse CARS spectroscopy of (CH₂Cl)₂ moleculesin the liquid phase. As can be seen, for the material with two resonantlevels, such as (CH₂Cl)₂, two resonant peaks (at 652 cm⁻¹ and 750 cm⁻¹)are observed in the Raman spectrum.

The spectral resolution of the Fourier transform operation is betterthan the pulse bandwidth by a factor of 40 (i.e., about 30 cm⁻¹). It islimited by the maximal number of phase modulation periods on the spatiallight modulator (SLM), technically determined by the number of pixels onthe SLM.

Thus, spectral resolution can be optimized by employing a simplesinusoidal phase function. Breaking the single pulse into a longertrain, than used in the above example, containing a larger number ofpulses can further reduce the nonresonant background, which dependsdirectly on the pulse peak intensity. This is achieved by adding higherharmonics orders to the applied phase functions. This phase function canbe expressed as a summation of the different harmonics orders as${{\Phi(\omega)} = {\sum\limits_{n}\quad{A_{n}{\cos\left( {C_{n}\omega} \right)}}}},$where A_(n) are the different harmonic order coefficients.

Referring to FIGS. 5A-5B, a demonstration of the nonresonant backgroundsuppression by using periodic spectral functions with additionalharmonics is illustrated for methanol (nonresonant component only) andCH₃I (a single resonance at 523 cm⁻¹), respectively. The CARS signal ofrelative intensity is shown versus the number of phase oscillationperiods across the spatial light modulator for both a sinusoidal phasefunction Φ(ω)=1.25 cos(Cω) (curve 51) and a phase function Φ(ω)=1.4cos(Cω)−1.4 cos(2Cω) which contains an additional harmonic component(curve 52). The CARS signal is plotted relative to that obtained by atransform-limited pulse (0 phase function oscillations). It should benoted that to achieve adequate suppression of the nonresonant backgroundat least several oscillation periods of the phase function across thepulse spectrum are necessary.

FIG. 5A shows how the use of a phase function containing only oneadditional harmonic allows for attenuating the nonresonant background bynearly two orders of magnitude.

FIG. 5B shows that the use of a phase function containing two componentsattenuates significantly the nonresonant background, while the resonantcomponent is almost completely restored. The achieved contrast betweenthe resonant signal and the nonresonant background is thus greatlyimproved.

According to another example of the phase control, the spectral phase ofthe excitation pulse can be controlled by applying to the excitationpulse a narrow-band phase gate near its short wavelength (high-energy)end. In other words, a narrow-band feature is applied to the pulse forinducing sharp changes in the phase of the factor E(ω−Ω) in Eqs. (1) and(2). Preferably, but not necessarily, the phase of the phase gatespectral function can be shifted by π at ω−Ω. Such spectral phasefunction hereinafter will be referred to as a π phase gate. For example,a bandwidth of the π phase gate can be in the range of about 0.5 nm to 3nm (i.e., 5-30 cm⁻¹). Preferably, the π phase gate is spectrally locatedin the vicinity of a short wavelength end of the excitation pulse.

In this scheme, a narrow spectral band in the excitation pulse is phaseshifted, serving as an effective probe, and the Raman spectrum isextracted from the interference pattern of the resonant signal with thenonresonant background.

Referring to FIGS. 6A-6C, there is illustrated an effect of a π phasegate on the temporal shape of the pulse and on the population amplitude,according to this specific example of the phase control.

FIG. 6A shows an example of the excitation pulse spectral intensity 61,a spectral phase 62 of the transform limited (unshaped) pulse, and a πphase-gate 63 of the shaped pulse. In this example, the π phase-gate 63has the bandwidth of about 1.5 nm centered at 790 nm. Also shown is atypical spectral region where the CARS signal can be measured. Thus, inorder to avoid the spectral overlap between the input pulse and a CARSsignal the power spectrum of the input pulse is blocked at about 780 nm.Note that the spectral intensity of the CARS signal is identified by areference numeral 64.

FIG. 6B shows in time domain a temporal intensity (temporal envelope) ofa transform limited pulse 65 and a temporal intensity of a π phase-gateshaped pulse 66. As can be seen, the narrow π phase gate hardly effectthe form of the pulse, merely reducing the peak intensity by about 15%.

FIG. 6C shows a calculated population amplitude A(Ω) for the transformlimited pulse 67 and for the pulse having the modulated phase 68. As canbe seen in FIG. 6C, for the case of the transform limited pulse, thepopulation amplitude decays monotonically versus the vibration energy.The changes due to the phase gate slightly modify the populationamplitude A(Ω). This modification depends on the width of the phasegate. Thus, the population amplitude A(Ω) is hardly modified for anarrow gate, since the energy content in a narrow spectral band part isnegligible compared with the entire pulse energy.

The resonant signal from a level Ω_(R) at any given frequency ω iscentered at ω−Ω_(R), due to a rather narrow band probe. In contrast, thenonresonant background signal is a coherent sum contributed by a largeportion of the pulse bandwidth. Thus, phase changes over a narrowspectral band, while dramatically affecting the phase of the resonantsignal, hardly modify the phase of the nonresonant signal. The relativephase between the nonresonant background signal and the resonant signalshould therefore vary rapidly, inducing either constructive ordestructive interference at the phase gate edges. Measuring the totalCARS spectrum, the interference pattern between the resonant signal andthe nonresonant background can be interpreted to reveal the vibrationalenergy level diagram.

The effect of the phase control by using an excitation phase with anarrow-band phase gate is demonstrated in the numerical simulationresults shown in FIGS. 7A-7D.

FIG. 7A shows a calculated CARS electric field as a function offrequency (spectrum) of both the resonant contribution 71 and thenonresonant contribution 72 along with the relative phase 73 betweenthem for a transform limited pulse illuminating of iodomethane (resonantat 523 cm⁻¹). FIG. 7B shows the calculated resulting CARS spectrum 74for the illuminating of iodomethane with a transform limited pulse.

As can be seen, the nonresonant background spectrum 72 monotonicallydecreases towards higher energies (short wavelengths), while theresonant signal 71 resembles the excitation pulse spectrum, shifted bythe Raman level energy. The relative phase 73 between the two is nearlyconstant at about π/2, rising to about π below 749 nm.

FIG. 7C shows a calculated CARS spectrum of both the resonantcontribution 75 and the nonresonant contribution 76 along with therelative phase 77 between them for the illuminating of iodomethane by aπ phase gate shaped pulse. FIG. 7D shows the calculated resulting CARSspectrum 78 for the transform limited pulse illuminating of iodomethane.Three effects can be seen in FIG. 7C. First, there is some reduction ofthe nonresonant background component. Second, the relative phase betweenthe resonant component and the nonresonant background component variesbetween 0 (constructive interference) at the high-energy (shortwavelength) side of the gate, and π (destructive interference) at thelow energy (long wavelength) side. Third, two new peaks 79 appear in theresonant spectrum 75, at both ends of the phase gate. The peaks 79 aredue to a transient enhancement effect demonstrated by Oron et al.previously in conventional multi-beam CARS and described in Phys. Rev.Lett., 2002, V. 88, P. 63004.

As can be seen in FIG. 7D, the net result is a sharp peak 710, followedby a deep dip 720 in the total CARS signal 78. It should be noted thatthis peak-dip feature determines the energy of the vibrational levelwith an accuracy of the phase gate width. For example, the phase gatecan be defined by three pixels on the SLM, corresponding, to about 25cm⁻¹, that is about 40 times better than the excitation pulse width.

Referring to FIGS. 8A and 8B, there are illustrated measured normalizedCARS spectra for the case of a transform limited pulse (curves 81 a and81 b) and the case of a phase gate shaped pulse (curves 82 a and 82 b),respectively, for methanol (having nonresonant component only) and foriodomethane (having a resonant at 523 cm⁻¹). The peak-dip feature due tothe resonant contribution at 523 cm⁻¹ can be seen in the iodomethanespectrum (FIG. 8B), while the normalized methanol spectrum (FIG. 8A)remains nearly unchanged.

The Raman level structure can be easily extracted from the measuredspectrum by considering, for example, the normalized spectral intensityvariation of the CARS signal $\begin{matrix}{{f(\Omega)} = {- \frac{{I\left( {\omega_{g} + \Omega - {\Delta/2}} \right)} - {I\left( {\omega_{g} + \Omega + {\Delta/2}} \right)}}{\int_{\omega_{g} + \Omega - {\Delta/2}}^{\omega_{g} + \Omega + {\Delta/2}}{{I(\omega)}\quad{\mathbb{d}\omega}}}}} & (7)\end{matrix}$where ω_(g) is the central frequency of the phase gate and Δ is the gatewidth. It should be understood that the normalization is required tocompensate for the decrease in the nonresonant background towards higherenergies.

Referring to FIGS. 9A-9D, plots of the normalized spectral intensityf(Ω) derived from the measured CARS spectra (curves 91, 92, 93 and 94)are given in for several materials, along with simulation predictions(curves 95, 96, 97 and 98) obtained by computer simulations.

As can be seen in FIG. 9A, nearly flat line is observed for methanol,having no Raman level in this range. The 459 cm⁻¹ level of carbontetrachloride is easily observed in FIG. 9B. For mesitylene (FIG. 9C),having two Raman levels at 515 cm⁻¹ and 575 cm⁻¹ two well-separatedpeaks can be seen. At the high-energy end, the 652 cm⁻¹ level of carbondisulfide is shown in FIG. 9D. It should be noted that the spectralresolution observed in these figures is of the order of about 30 cm⁻¹,(i.e., almost a factor of 40 better than the excitation pulsebandwidth). The resolution is determined by both the width of the phasegate (25 cm⁻¹) and the monochromator resolution (about 8 cm⁻¹). Theminimal phase gate width is determined by both the pixellization of theSLM and by the spot size of the incident beam on it. In all the examplespresented above the phase gate consisted of three pixels on the SLM.

It should be understood that for detection of a given Raman level, it ispossible to control the relative intensity ratio between the resonantand the nonresonant components by varying the spectral location of thephase gate. This is due to the fact that the nonresonant backgrounddecreases towards higher energies. Additionally, a further control ispossible by varying the phase gate width. Thus, a wider probe width canimprove the resonant to nonresonant intensity ratio (while resulting inlower spectral resolution). In this case, even weak Raman levels can beobserved by using this scheme.

The benefits of broadband excitation can be fully exploited whenattempting to detect materials with several vibrational bands in themeasured energy range. In this case, a spectral phase mask havingmultiple phase gates at appropriate locations can be used to generate alarge coherent spectral feature in the CARS spectrum, due to theconstructive interference of the resonant contributions from the variouslevels.

According to another embodiment of the invention, the CARS process iscontrolled by controlling the polarization of the excitation pulse. Itshould be appreciated that the polarization control can be carried outin addition to the shaping, i.e., correcting of the dispersion of theinput pulse and assigning of the desired phase to each frequencycomponent of the driving pulse.

In particular, the polarization control can be used to break theultrashort input pulse into a broadband pump and a narrow-band probewith orthogonal polarizations.

The nonlinear polarization producing the CARS signal driven by anelectric field whose spectrum is E(ω) can be approximated fornonresonant transitions: $\begin{matrix}{{P_{{nr}{(j)}}^{(3)} \propto {\chi_{jklm}^{nr}{\int_{0}^{\infty}\quad{{\mathbb{d}\Omega}\quad{E_{k}\left( {\omega - \Omega} \right)}{A_{lm}(\Omega)}}}}},} & (8)\end{matrix}$where A_(lm)(Ω)=∫₀ ^(∞)dω′E_(l)*(ω′−Ω)E_(m)(ω′) is the populationamplitude and χ_(jklm) ^(nr) is the third order susceptibility tensor.

In turn, for a singly resonant Raman transition through an intermediatelevel |i

at an energy of

Ω_(R) and a bandwidth Γ one can obtain: $\begin{matrix}{P_{r{(j)}}^{(3)} \propto {\chi_{jklm}^{nr}{\int_{0}^{\infty}\quad{{\mathbb{d}\Omega}\frac{E_{k}\left( {\omega - \Omega} \right)}{\left( {\Omega_{R} - \Omega} \right) + {i\quad\Gamma}}{{A_{lm}(\Omega)}.}}}}} & (9)\end{matrix}$

The two main differences between the resonant and nonresonant componentsare as follows. First, the resonant component has a narrow spectralresponse, centered at Ω=Ω_(R), whereas the spectral response of thenonresonant component is broad. Second, the response of the resonantcomponent inverts sign at about Ω=Ω_(R), while the nonresonant responsehas a constant phase. It will be shown hereinbelow how these differencesare used to reduce the nonresonant component while enhancing theresonant component.

One approach to single-pulse polarization controlled CARS would be torotate by π/2 the polarization of the excitation pulse in a narrow bandat its high energy end, from an x-plane to a y-plane, and to monitor theCARS signal in the y-plane. As a result, the monitored signal willeffectively dependent only on A_(xx) (as the polarization field), and onE_(y) (as the probe). This is true for both the resonant and nonresonantterms.

As was mentioned above for the case of the coherent control by means ofa phase gate, the ratio of the resonant signal to the nonresonant signalof the background as well as the spectral resolution can be improvedwhen the probe pulse becomes longer. According to this embodiment of theinvention, the duration of the probe pulse is determined by the spectralwidth of the polarization shifted band.

Referring to FIGS. 10A-10D, examples of the CARS spectra fromiodomethane obtained with polarization-only shaping are plotted forvarious polarized probe spectral bandwidths. The total bandwidth of thepolarized probe decreases as follows: 4.5 nm (for the example shown inFIG. 10A); 2.3 nm (for the example shown in FIG. 10B); 1.2 nm (for theexample shown in FIG. 10C) and 0.6 nm (for the example shown in FIG.10D). As the probe bandwidth is decreased from 4.5 nm (corresponding to400 fs) to 0.6 nm (corresponding to 3 ps, the resonant component, due toits narrow spectral response, becomes narrower but maintains itsstrength. In contrast, the nonresonant background component, having abroad spectral response, becomes weaker but maintains its spectralshape. Since the two are coherent, they generate an interferencepattern, interfering constructively at the low-energy end of the probepulse, and destructively at its high-energy end. This interferencepattern obscures the interpretation and calls for further reduction ofthe nonresonant background.

Further reduction of the nonresonant background is achieved by usingboth the polarization control and the phase control. It will be shownbelow that the combination of both the phase and the polarizationcontrols can lead to nearly complete suppression of the nonresonantcomponent, yielding background-free single-pulse multiplex CARS spectrawith a high spectral resolution.

FIG. 11 exemplifies the spectral intensity of a phase and polarizationshaped excitation pulse. According to this example, a π phase-shiftedgate 110 is introduced at a y polarization shifted band 111, serving asa probe. The probe is thus split into two spectrally distinct longerprobe pulses with opposite phase. Due to the broad nonresonant spectralresponse, the nonresonant background from these two probe pulsesinterferes destructively. Since the A_(xx)(Ω) component of the amplitudeis a very smooth function, these two probe pulses are almost equal inmagnitude. As a result, the nonresonant background component of the CARSsignal can be reduced by orders of magnitude.

This reduction can be alternatively viewed in time domain. FIG. 12 showsa schematic drawing of the electric field envelope versus time in boththe x polarization (curve 121) and the y polarization (curve 122) forboth phase and polarization shaped pulse. For convenience, the xpolarization field has been reduced by about two orders of magnitude. Ascan be appreciated, the π phase gate modifies the temporal shape of they polarized probe so that the electric field envelope crosses zero atthe peak of the x polarized driving field.

Due to the instantaneous nonresonant response, the nonresonantbackground is almost completely suppressed. The resonant signal responseis different. It should be noted that the π phase gate compensates forthe sign inversion of the denominator in Eq. 9, leading to an increasedresonant signal over a narrow spectral band shifted by the Raman levelenergy from the π phase gate location.

Referring to FIGS. 13A-13C, examples of the CARS spectra fromiodomethane obtained with both polarization and π phase gate shaping areplotted for various probe spectral bandwidths. According to thisexamples, the total probe bandwidth varies from 4.5 nm (for the exampleshown in FIG. 13A) to 2.4 nm (for the example shown in FIG. 13B), andthen further to 1.2 nm (for the example shown in FIG. 13C). When themeasured spectra obtained by using the polarization and phase shapedprobe pulses (shown in FIGS. 13A-13C) are compared to the measuredspectra obtained by using polarization-only shaped transform limitedpulses (shown in FIGS. 10A-10D), a dramatic decrease in the nonresonantbackground can be seen. It should be noted that the small nonresonantbackground that is still observed using the phase-shaped probe is infact a small fraction (about 0.05%) of the χ_(xxxx) component which“leaks” through the polarizer due to small birefringence of themicroscope objective and collection optics. However, since thisbackground component is independent on the applied phase andpolarization, it can be easily subtracted.

Referring to FIGS. 14A-14C, examples of Raman spectra of several simplemolecules obtained with phase and polarization shaped pulses areillustrated. The total probe bandwidth has been about 1.2 nm, includinga π phase gate at the bandwidth's center. The “raw” measured CARSspectra are shown in the left part of the figures. It should be notedthat the small nonresonant background that is still observed is in facta small fraction (about 0.05%) of the χ_(xxxx) component which “leaks”through the polarizer due to small birefringence of the microscopeobjective and collection optics. Since this background component isindependent on the applied phase and polarization, it can be easilysubtracted. The extracted Raman spectra, from which the background dueto birefringence was subtracted, are plotted on the right part of thefigures.

FIG. 14A shows a peak 141 corresponding to the 523 cm⁻¹ Raman level ofiodomethane. The full-width at half maximum of this peak is about 15cm⁻¹.

The measured Raman spectrum of 1,2-dichloroethane is shown in FIG. 14B.The levels at 652 cm⁻¹ and 750 cm⁻¹, separated by 98 cm⁻¹ are seen astwo very well separated peaks 142 and 143. This spectrum also has a peak144 corresponding to the level at 298 cm⁻¹ located at the lower limit ofthe detectable region.

The Raman spectrum of p-xylene, having a peak 145 corresponding to thelevel at 830 cm⁻¹ is shown in FIG. 14C. This example demonstrates theability of the technique of the present invention to observe thehigh-energy end of the detectable region. It should be noted that themeasured energy range can be extended to higher frequencies (1000-15000cm⁻¹) by using shorter pulses.

Single-pulse CARS is particularly suitable for nonlinear microscopy.FIG. 15 exemplifies a single-pulse CARS microscope 150 according to theinvention. Generally, the CARS microscope 150 includes all the elementsof the single-pulse spectrometer (20 in FIG. 2A or 200 in FIG. 2B)needed for inducing a CARS process in the molecules of a target materialand for detecting the CARS signal scattered by the material. Morespecifically, the CARS microscope 150 includes means for inducing theCARS process by producing a beam constituted of unitary opticalexcitation pulses and directing it through the target material placed onthe sample holder 29. According to the technique of the presentinvention, each pulse is a unitary pulse that carries a pump photon, aStokes photon and a probe photon. The CARS microscope thus includes alaser 21 adapted for generating at least one transform limited opticalpulse and a programmable pulse shaper 22 (constituting a control meansfor coherently controlling the CARS process) operable for shaping thetransform limited optical excitation pulse obtained from the laser 21, adetector unit 26, and a light directing optics. The shaping is carriedout by correcting the pulse dispersion as well as assigning the desiredphase (and, optionally, polarization) to the pulse, as described above.

The light directing optics of the CARS microscope 150 includes afocusing assembly (e.g., a microscope objective) 24 a arranged forcreation a focal spot 152 formed by the beam on the target material; alens assembly 24 b arranged for collecting the output CARS signal fromthe target material; and a filtering assembly 25 operable for filteringthe collected output CARS signal propagating towards the detector unit26. All these components are similar to those described above inconnection with the CARS spectrometer system (20 in FIG. 2A or 200 inFIG. 2B).

The CARS microscope 150 utilizes scanning of at least a portion of thetarget material with the focal spot 152, which can be implemented bysupporting the sample holder on a stage 151 driven for movement, and/orby mounting at least some of optical elements for movement with respectto the sample holder to thereby appropriately deflect the incident beam.The microscope objective 24 a may for example be that commerciallyavailable from ZEISS. The stage driver may include a piezoelectrictransducer, e.g., P-282 XYZ Nano positioners commercially available fromPhysik Instrumente (PI) GmbH.

An example of single-pulse spectrally resolved microscopy isdemonstrated herein below. A selected target material was a glasscapillary plate with 10-μm holes filled with CH₂Br₂ (having a resonantat 577 cm⁻¹). The sample was raster-scanned around the focused laserbeam using computer-controlled piezoelectric drivers.

An image shown in FIG. 16A was taken with a pulse shape maximizing therelative intensity of the resonant contribution, whereas the image shownin FIG. 16B was taken with a pulse shape minimizing this intensity.

The predominantly resonant signal from the filled holes shown in FIG.16A is larger by a factor of 4 than that in FIG. 16B, while the signalsobtained from the glass is almost similar.

FIG. 16C shows an image that is a difference between the images in FIG.16A and FIG. 16B, depicting the signal from the 577 cm⁻¹ vibrationallevel of CH₂Br₂. This image appears inverted relative to that obtainedby using a transform-limited pulse (shown in FIG. 16D), where the glass,having a larger nonresonant signal, appears brighter. The image in FIG.16C demonstrates the ability of the microscope to spectrally resolve theRaman resonant contribution of a single vibrational level. In apractical system, the difference image can be directly measured bylock-in detection, alternating the phase masks of the images shown inFIGS. 16A and 16B at a high frequency.

It should be appreciated that for materials having more than onevibrational level it is possible to improve the detection selectivity bytailoring shaped pulses to induce constructive quantum interference ofthese levels.

As such, those skilled in the art to which the present inventionpertains, can appreciate that while the present invention has beendescribed in terms of preferred embodiments, the concept upon which thisdisclosure is based may readily be utilized as a basis for the designingof other structures, systems and processes for carrying out the severalpurposes of the present invention.

Although the example of utilization of the CARS spectrometer techniqueof the present invention were shown for CARS microscopy, the spectralmeasurement utilizing the coherent control of the present invention canbe easily combined with other nonlinear microscopic methods such asmultiphoton fluorescence and third-harmonic generation using the samemicroscope system.

Also, it is to be understood that the phraseology and terminologyemployed herein are for the purpose of description and should not beregarded as limiting.

It is important, therefore, that the scope of the invention is notconstrued as being limited by the illustrative embodiments set forthherein. Other variations are possible within the scope of the presentinvention as defined in the appended claims and their equivalents.

1. A method for use in inducing a Coherent Anti-Stokes Raman Scattering(CARS) signal from a medium, the method comprising shaping a unitaryoptical pulse carrying a pump photon, a Stokes photon and a probe photonand a producing a unitary optical excitation pulse, said shapingcomprising blocking a spectrum of the unitary optical pulse in anexpected spectral range of the CARS signal, the method thereby enablinginducing and detection of the CARS signal from the medium by applicationof said unitary optical excitation pulse to the medium.
 2. The method ofclaim 1, wherein said shaping of the unitary optical pulse comprisesassigning a desired phase to each wavelength component of the unitaryoptical pulse, said assigning comprises modulating a spectral phase ofthe unitary optical pulse by using a desired spectral phase function,the spectral phase function being determined by a closed feedback loop,said shaping thereby producing said unitary optical excitation pulse. 3.The method of claim 1, wherein said shaping of the unitary optical pulsecomprises assigning a desired phase to each wavelength component of theunitary optical pulse, said assigning comprises modulating a spectralphase of the unitary optical pulse by using a desired spectral phasefunction changing a symmetry of a polarization defining productE(ω−Ω)A(Ω) for different values ω around Ω=Ω_(R), E(ω) being a spectrumof an electric field of the excitation pulse, A(Ω) being a probabilityamplitude to populate a vibrational level with energy

Ω,

Ω_(R) being an energy of an intermediate level of a CARS transition. 4.The method of claim 1 comprising producing said unitary optical pulse.5. The method of claim 4, said producing comprising providing atransform limited optical pulse.
 6. The method of claim 1, wherein theblocking is applied to wavelengths shorter than a long edge of theexpected spectral range of the CARS signal.
 7. The method of claim 6wherein said desired spectral phase function is a periodic function. 8.The method of claim 7 wherein said desired spectral phase function isprovided with an involvement of at least one phase gate having abandwidth substantially narrower than a bandwidth of the unitary opticalexcitation pulse.
 9. The method of claim 8 wherein at least one phasegate is a π phase gate.
 10. The method of claim 9 wherein the bandwidthof said π phase gate is in a range of about 0.5 nm to 3 nm.
 11. Themethod of claim 9 wherein said π phase gate is spectrally located withina short wavelength part of the unitary optical excitation pulse.
 12. Themethod of claim 1 wherein said shaping comprises passing the unitaryoptical pulse through a Spatial Light Modulator (SLM).
 13. The method ofclaim 1 wherein the shaping comprises splitting said unitary opticalpulse into a broadband pump component and a narrow-band probe componenthaving substantially orthogonal polarizations.
 14. The method of claim13 wherein said broadband pump and narrow-band probe components arephase-controlled.
 15. The method of claim 1 wherein said unitary opticalpulse is in a range of about 5 to 100 femtoseconds.
 16. A system for usein inducing a Coherent Anti-Stokes Raman Scattering (CARS) signal from amedium, the system comprising a pulse shaper for accommodating in anoptical path of a unitary optical pulse carrying a pump photon, a Stokesphoton and a probe photon and being configured and operable to shape theunitary optical pulse into a unitary optical excitation pulse, the pulseshaper comprising a block element for blocking a spectrum of the unitaryoptical pulse in an expected spectral range of the CARS signal, and aprogrammable shaping assembly operable to modulate a spectral phase ofthe unitary optical pulse by a desired spectral phase function andassign a desired phase to each of the unitary optical pulse wavelengthcomponents.
 17. The system of claim 16 comprising a closed feedback loopdetermining said spectral phase function.
 18. The system of claim 16,wherein said spectral phase function changes a symmetry of apolarization defining product E(ω−Ω)A(Ω) for different values ω aroundΩ=Ω_(R), E(ω) being a spectrum of an electric field of the excitationpulse, A(ω) being a probability amplitude to populate a vibrationallevel with energy

Ω,

Ω_(R) being an energy of an intermediate level of a CARS transition. 19.The system of claim 16 comprising a single laser operable to generate atleast one unitary optical pulse.
 20. The system of claim 19, said singlelaser being operable to generate a transform limited optical pulse. 21.The system of claim 19 wherein said single laser is a Ti:Sapphire laser.22. The system of claim 16 comprising a detector unit for receiving anoutput CARS signal produced by the medium excited by said unitaryoptical excitation pulse and generating data indicative thereof.
 23. Thesystem of claim 22 wherein said detector unit includes a lock-inamplifier.
 24. The system of claim 22, comprising light directing opticsfor directing the unitary optical excitation pulse to the medium anddirecting the output CARS signal to the detector unit.
 25. The system ofclaim 16 wherein said programmable pulse shaping assembly comprises aninput dispersive device for spatially separating wavelength componentsof the unitary optical pulse; a programmable Spatial Light Modulator(SLM) accommodated in the optical path of said separated wavelengthcomponents and operable to modulate said separated wavelength componentsby at least the desired spectral phase function; and an outputdispersive device for recombining the modulated wavelength componentsinto the unitary optical excitation pulse.
 26. The system of claim 16,wherein said programmable pulse shaping assembly comprises apolarization control assembly operable to apply a polarization rotationto predetermined wavelength components of the unitary optical pulse andthereby produce a broadband pump component and a narrow-band probecomponent having substantially orthogonal polarizations, and to apply across polarization filtering to a signal propagating from the medium toa detector unit for extraction of the cross-polarized CARS signal. 27.The system of claim 26, wherein said polarization control assemblycomprises an input polarization filtering unit accommodated in theoptical path of the unitary optical pulse.
 28. The system according toclaim 26, wherein the polarization control assembly comprises a SpatialLight Modulator (SLM) arrangement.
 29. The system of claim 16comprising: a filtering assembly accommodated in the optical path of anoutput CARS signal propagating from the medium towards a detector unit,said filtering assembly being operable to apply a frequency filtering tosaid output CARS signal.
 30. The system of claim 29 wherein saidfiltering assembly includes at least one of the following: a bandpassfilter, a short-pass filter; a spectrograph.
 31. The system of claim 29wherein said filtering assembly includes a monochromator.
 32. The systemof claim 16 wherein the unitary optical pulse is in a range of about 5to 100 femtoseconds.
 33. The system of claim 16 wherein said desiredspectral phase function is a periodic function.
 34. The system of claim16 comprising at least one phase gate having a bandwidth substantiallynarrower than the bandwidth of the unitary optical excitation pulse. 35.The system of claim 34 wherein the phase gate is a π phase gate.
 36. Thesystem of claim 35 wherein the bandwidth of said π phase gate is in arange of about 0.5 nm to 3 nm.
 37. The system of claim 35 wherein said πphase gate is spectrally located within a short wavelength part of theexcitation pulse.
 38. A CARS spectrometer comprising the system of claim16.
 39. A CARS microscope comprising the system of claim 16.